Drug‐Primed Self‐Assembly of Platinum‐Single‐Atom Nanozyme to Regulate Cellular Redox Homeostasis Against Cancer

Abstract Single‐atom nanozymes (SAzymes) with high catalytic activity exhibit the potential to disequilibrate the reactive oxygen metabolic balance in the tumor microenvironment (TME), which contains several endogenous reductive substances such as glutathione (GSH). Herein, a novel nano‐assembly (CDs@Pt SAs/NCs@DOX) is first constructed using drug‐primed platinum (Pt) single‐atom or nanocluster nanozymes with a Pt loading of 34.8%, which exhibits prominent dual enzymatic activities to mimic peroxidase (POD) and glutathione oxidase (GSHOx). The unique GSHOx‐like activity can efficiently scavenge GSH with a relatively low K m (1.04 mm) and high V max (7.46 × 10−6 m s−1), thus avoiding single oxygen (1O2) depletion. CDs@Pt SAs/NCs@DOX simultaneously demonstrates low‐temperature photothermal therapy and TME‐ or laser‐controlled disassembly and drug release, which can effectively regulate cellular redox homeostasis and achieve high tumor growth inhibition. These outcomes may provide promising strategies for the preparation of Pt SAzymes with multiple activities and variable‐sized nano‐assemblies, allowing for broader applications of SAzymes and nano‐assemblies in the biomedical field.


Experimental Section
Fluorescence quantum yield (FQY) measurements: The FQY of CDs@Pt SAs/NCs was obtained by the comparative method, which is calculated using the slope of the line relation between the integrated fluorescence intensities and absorbance.In this case, FQY can be calculated using the following equation: where m and n are the slope of the linearity obtained from the plot of the absorbance against the integrated fluorescence intensities and the refractive index of solvent, respectively.R represents the reference fluorophore of known FQY.Fluorescein with FQY of 79% in 0.1 M NaOH was chosen as the reference.The refractive indexes of 0.1 M NaOH and ultrapure water are 1.33, (n 2 /n R 2 ) will be 1, so the FQY can be obtained from the quotient of the two slopes.with the lattice fringe spacing of 0.12 nm and 0.15 nm, corresponding to the (311) and (220) plane of face-centered cubic (fcc) phase of Pt, [1,2] respectively.and h) CDs@Pt SAs/NCs at excitation and absorption wavelengths of 339 nm.CDs@Pt SAs/NCs has a weak absorption peak at 324-377 nm that is obviously different from that of CDs and H 2 PtCl 6 , which may be a new absorption peak produced after the reaction of CDs and H 2 PtCl 6 .A new wide absorption peak at 500-900 nm can be found in the absorption spectrum of CDs@Pt SAs/NCs, indicating that CDs@Pt SAs/NCs have been successfully prepared and have photothermal potential.Under different excitation wavelengths, the emission wavelengths of CDs produce a redshift from 443 nm to 472 nm, while the emission wavelengths of CDs@Pt SAs/NCs hardly change between 436 nm and 440 nm, illustrating that CDs and CDs@Pt SAs/NCs have the excitation-dependent and excitation-independent photoluminescence (PL) behaviors, respectively, which may be attributed to the change of the optical properties of CDs caused by Pt SAs/NCs.Using quinine sulfate as reference, the FQYs of CDs and CDs@Pt SAs/NCs were calculated to be 33.5% and 3.05%, respectively.
The high FQY of CDs may be caused by the surface-state and abundant amino functional groups, while the low FQY of CDs@Pt SAs/NCs may be attributed to the change of the surface-state and functional groups of CDs after the reduction of Pt SAs/NCs to the surface of CDs, resulting in the significant fluorescence attenuation of CDs.CDs@Pt SAs/NCs@DOX, indicating the formation of CDs@Pt SAs/NCs@DOX and confirming the successful loading of the driving DOX molecules onto CDs@Pt SAs/NCs.
At an excitation wavelength of 339 nm, the emission peaks of CDs@Pt SAs/NCs and DOX were 436 and 596 nm, respectively, while the emission peaks of CDs@Pt SAs/NCs@DOX were 432 and 596 nm.At an excitation wavelength of 482 nm for CDs@Pt SAs/NCs@DOX, only the emission peak of DOX at 596 nm can be found.In essence, both CDs@Pt SAs/NCs and DOX moieties in CDs@Pt SAs/NCs@DOX can maintain their own PL characteristics.
The release of DOX in CDs@Pt SAs/NCs@DOX reached equilibrium after 6.5 h, and the release rates of DOX at pH 6.5, 7.0, and 7.4 were 74%, 29%, and 10%, respectively, suggesting that the release of DOX was controlled by pH and was much higher in a weakly acidic environment than in a neutral environment.After four cycles of 808 nm laser irradiation, the release rate of DOX increased from 29% to 48% at pH 7.0, illustrating that laser irradiation not only increased the temperature of CDs@Pt SAs/NCs@DOX, but also promoted the release of DOX from CDs@Pt SAs/NCs@DOX.SAs/NCs@DOX and TMB under a fixed H 2 O 2 concentration.h) PL spectra of CDs@Pt SAs/NCs@DOX (0.5 mg mL -1 ) with different concentrations of GSH.When the concentration of CDs@Pt SAs/NCs@DOX, reaction temperature, reaction time, and pH were 300 μg mL -1 , 45 o C, 30 min, and 4.0, respectively, CDs@Pt SAs/NCs@DOX can produce the most 1 O 2 and reveal the best POD-like activity.The V max of CDs@Pt SAs/NCs@DOX for H 2 O 2 and TMB were 5.71×10 -8 M s -1 and 6.21×10 -8 M s -1 , respectively.The K m of CDs@Pt SAs/NCs@DOX for H 2 O 2 and TMB were 234.39 mM and 0.86 mM, respectively.(horseradish peroxidase); b) (porphyrin-like single Fe sites on N-doped carbon nanomaterials); c) (the monodispersed ZIF-8 derived carbon nanospheres containing zinccentered porphyrin-like structure); d) (Fe-N-C single-atom nanozyme); e) (Pt single atoms or nanoclusters (Pt SAs/NCs) on the surface of CDs); f) (a nano-assembly using DOX-primed CDs@Pt SAs/NCs).The V max of CDs@Pt SAs/NCs@DOX for H 2 O 2 and TMB were greater than HRP, indicating that CDs@Pt SAs/NCs@DOX had a faster catalytic reaction rate.The K m of CDs@Pt SAs/NCs@DOX for H 2 O 2 and TMB were higher than CDs@Pt SAs/NCs, indicating the weaker affinity between CDs@Pt SAs/NCs@DOX and substrates, which may be due to the fact that part of Pt was wrapped during the formation of CDs@Pt SAs/NCs@DOX.
Table S2.The V max and K m of CDs@Pt SAs/NCs and other nanozymes with GSH as the substrate for GSHOx-like catalysis.

Figure S1 .
Figure S1.Characterization of CDs@Pt SAs/NCs.a) Element mapping and b) high-angle annular dark-field scanning transmission electron microscopy (HAADF-STEM) images of CDs@Pt SAs/NCs.The uniform distribution of C, O, N, and Pt elements can be found in element mapping, indicating that Pt has been successfully reduced to the surface of CDs.The HAADF-STEM image illustrates that Pt exists as single atoms (A: yellow circle) and nanoclusters (B: faced Pt clusters; C: highly disordered clusters; D: strained lattices of Pt)

Figure S3 .Figure S4 .
Figure S3.TGA spectrum of CDs@Pt SAs/NCs.The TGA curve was obtained by treating CDs@Pt SAs/NCs in a nitrogen (N 2 ) atmosphere with a heating rate of 10 o C min -1 .The residual weight of CDs@Pt SAs/NCs is about 32.67% when the temperature is higher than 900 o C, which is mainly inorganic Pt in CDs@Pt SAs/NCs.

Figure S5 .
Figure S5.Characterization of CDs@Pt SAs/NCs@DOX.a) The zeta potentials and b) UVvis absorption spectra of DOX, CDs@Pt SAs/NCs, and CDs@Pt SAs/NCs@DOX.c) The emission spectra of CDs@Pt SAs/NCs, DOX, and CDs@Pt SAs/NCs@DOX under different excitation wavelengths.d) TGA curve of CDs@Pt SAs/NCs@DOX.The characteristic absorption peaks of 225, 290, and 500 nm of DOX appeared in the absorption spectrum of

Figure S10 .
Figure S10.The photothermal effect of CDs@Pt SAs/NCs and CDs@Pt SAs/NCs@DOX.a) Temperature changes (ΔT) of CDs@Pt SAs/NCs during six on/off cycles upon the 808 nm laser irradiation.b) Linear time data obtained from the cooling period.Inset: Heating and cooling curves of CDs@Pt SAs/NCs.c) Photothermal curves of CDs@Pt SAs/NCs@DOX with various concentrations irradiated by the 808 nm laser (0.37 W cm -2 ) at different times.( * p < 0.05, ** p < 0.01, *** p < 0.001, n = 3).d) Temperature changes (ΔT) of CDs@Pt SAs/NCs@DOX during six on/off cycles upon the 808 nm laser irradiation.e) The

Table S1 .
The maximum reaction rate (V max ) and Michaelis-Menton constant (K m ) of CDs@Pt SAs/NCs, CDs@Pt SAs/NCs@DOX, natural HRP, and other SAzymes with TMB and H 2 O 2 as the substrate for POD-like catalysis.